KR100455905B1 - Monascus purpureus KLHS4 having high pigment productivity - Google Patents

Abstract

The present invention relates to a novel Monascus purpureus KLHS4 having a high ability to produce red pigment, and more specifically, to use a Monascus persperus KLHS4 having a high ability to produce red pigment, and the Monascus persperus KLHS4. It relates to a manufacturing method. Monascus Perus KLHS4 of the present invention produces a large amount of red yeast pigment and antimicrobial substances compared to Monascus perperus (ATCC 16365), a kind of red yeast fungus, which is useful for glycosylation in the production of food additives such as food additive pigment, red wine and red rice. Can be used.

Description

Monascus purpureus KLHS4 having high pigment productivity}

The present invention relates to a novel Monascus purpurus KLHS4 having a high redness pigment production ability, and more specifically, to produce a red pigment using Monascus perus KLHS4 having a high redness pigment production capacity and the Monascus perperus KLHS4. It is about how to.

The red yeast bacterium belongs to the red yeast genus (Monascaceae) of the hematopoietic bacillus (Hemiascomycetaceaw) and is close to the yellow fungus. Currently, about 20 species and about 70 strains have been isolated and identified. The strain has been used mainly in southern China or Taiwan since 600 years, and because it produces red pigment, it is generally called red yeast bacteria, and red solid culture obtained by fermenting red yeast bacteria to grains This is called.

Hongguk's efficacy is described in the Chinese Ming Dynasty's classic text, `` It is easy to relieve weakness and no toxicity, it is useful for controlling indigestion and diarrhea, and promotes blood circulation and strengthens digestive function. It has been used as a medicine.

Monascus genus of red yeast rice has been used for the production of fermented foods such as red wine and red rice, and recently, red yeast pigment is used as a food additive. In particular, red yeast pigment has been known to have an antimutagenic effect that degrades the heterocyclic amines produced when cooking foods rich in protein (Izawa et al., J. Agric. Food Chem . 1997, 45). : 3980-3984; Watanabe et al., Mutat. Res. 1999, 444: 75-83). In addition, Monascus red yeast strains contain substances such as lovastatin or Monacolin, which inhibit the synthesis of cholesterol (Heber et al., Am. J. Clin. Nutr . 1999, 69: 231). It is used as a cholesterol lowering agent. It also produces citrinin-like substances such as Monascidin A, which are antibacterial, depending on the strains of red yeast rice (Blanc et al., Int. J. Food Microbiol . 1995, 27: 201- 213) Also used as an additive for antibiotics in food (Pak Si-Yong et al. , Kor. J. Appl. Microbiol. Biotechnol. 1999, 27: 172-178). In addition, it is also known that the water-soluble extract of red yeast fungus has an effect of strengthening blood pressure (Kushiro et al. , Nippon Jinzo Gakkai Shi . 1996, 38 (12): 625-633), and the usefulness of red yeast fungus is further expanded. to be.

In the meantime, various patents have been filed in connection with such red yeast bacteria, and Korean Patent Application No. 92-019729 (Monobacterial Monascus anka 732Y3 (KCCM10014)) has a red yeast pigment of Monascus anca, especially red. A variant strain of Monascus anka KFCC11832, which has a high pigment production amount, is described. Korean Patent Application No. 92-019730 (Method for producing a red yeast pigment by microorganisms) is used for culture medium from various kinds of red yeast strains. It describes a method for producing red yeast pigment by adding rice flour and peptone. In addition, Korean Patent Application No. 90-022510 (hongguk pigment extraction method) describes a method of extracting a pigment from monascus anchor belonging to the genus Monascus using ethanol as an extraction solvent.

Accordingly, the present inventors completed the present invention by separately providing a novel Monascus perus KLHS4, which produces a large amount of red pigment and antibacterial substances than the existing Monascus perus.

It is an object of the present invention to provide a novel Monascus Perus KLHS4 strain with high red pigment production.

1 is a photograph showing the growth type in potato-sugar-agar solid medium of the Monascus perus KLHS4 strain of the present invention,

A: Monascus perus KLHS4 front, B: Monascus perus KLHS4 rear,

C: front of the Monascus pertus ATCC16365,

D: Rear view of the Monascus Perus ATCC16365,

Figure 2 is a photograph showing the chromaticity of the pigment solution extracted with methanol from the culture cultured Monascus Perus KLHS4 strain in potato-sugar-agar solid medium,

I: Monascus Perus KLHS4,

II: Monascus Perus ATCC16365,

Figure 3 is a picture of the pigment spectrum by using a spectrometer (spectrometer) from the solid and liquid culture of the Monascus Perus KLHS4 strain,

A: methanol extract of potato-sugar-agar solid medium,

B: culture supernatant in potato-sugar liquid medium,

C: methanol extract from mycelium cultured in potato-sugar liquid medium,

Dotted line: Monascus perusus KLHS4,

Solid line: Monascus Perus ATCC16365,

Figure 4 is a photograph showing the pigment type of Monascus Perus KLHS4 strain by performing thin layer chromatography (TLC),

A: TLC visible light photo, B: TLC long wavelength UV photo,

PDB: potato-sugar liquid culture supernatant,

MY: potato-sugar liquid medium culture mycelia methanol extract,

PBA: potato-sugar-agar solid medium culture medium methanol extract,

I: Monascus Perus KLHS4,

II: Monascus Perus ATCC16365,

Figure 5 is a photograph showing the antibacterial action against Bacillus subtilis strain of Monascus Perus KLHS4 strain.

A: antibacterial ring seen from above, B: antibacterial ring seen from behind

I: Monascus Perus KLHS4,

II: Monascus Perus ATCC16365

In order to achieve the above object, the present invention provides a novel Monascus purpureus KLHS4 strain with a high red yeast pigment production capacity.

In addition, the present invention provides a method for producing a red yeast pigment using the Monascus Perus KLHS4.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Monascus Perus KLHS4 having a high red yeast pigment production capacity.

In the present invention, in order to select a novel Monascus perforus strain having a high red yeast pigment production capacity, by analyzing the pigmentation degree of the solid medium to screen a novel Monascus perforus strain with a high yield of red yeast pigment compared to other red yeast strains It was. The present inventors named the novel novel Monascus perperus strain having high red yeast pigment production ability as above, and named it as "Monascus perperus KLHS4", and deposited it on November 19, 2001 at the Korea Biotechnology Research Institute Gene Bank. Number: KCTC 18097P)

Monascus perusus KLHS4 of the present invention has lipolytic, predegradable, proteolytic and beta-glucosidase (β-glucosidase) activity. The length of the hyphae observed under the light microscope was varied and its width was 4 ± 0.5 um. A spore (conidium) is an oval shape separated from the hyphae and the septum in the form of a bulging front end of the mycelia branched from the parent hyphae. There is also Cleistothecium with oily spores. The red pigment was uniformly distributed in the cytoplasm of mycelia, and the optical microscopic findings of KLHS4 were similar to those of ATCC16365.

In order to examine the pigment spectrometer of the Monascus perus KLHS4 of the present invention, when compared with the Monascus perforus ATCC16365, which is known to have a high production of red pigment, the present invention, the Monascus perforus KLHS4 is a solid red pigment. The secretion into the medium showed a darker dark red color than the control of the medium, Monascus Perus ATCC16365 (see Fig. 1 ), and the color of the Monascus Perus KLHS4 mycelium was uniformly red, whereas the control of the control, Monascus Perus ATCC16365, was pink. I could see that. Therefore, it can be seen that the novel Monascus perforus KLHS4 of the present invention is a red yeast strain forming a uniform red mycelium.

In addition, after culturing the culture medium of the Monascus perus KLHS4 of the present invention finely immersed in methanol to extract the pigment and compare the color of the methanol extract solution, the Monascus perus KLHS4 of the present invention is darker than the control ATCC16365 exhibit red (see a in Fig. 2), a Pseudomonas coarse puffer Russ KLHS4 of the present invention from which it can be seen that the strain which produces a large amount of pigment as compared to Pseudomonas coarse puffer Russ ATCC16365 (see Fig. 3a).

From the above results, the Monascus perusus KLHS4 of the present invention can be usefully used for producing fermented foods such as red wine or red rice because of its high red pigment production.

In addition, the present invention provides a method for producing a red yeast pigment using the Monascus Perus KLHS4.

The red yeast pigment manufacturing method of the present invention

1) culturing Monascus perus KLHS4 in potato-sugar solid medium or liquid medium; And

2) The cultured solid medium or liquid medium consists of extracting the red pigment red with methanol.

In the medium of step 1, it is preferable to incubate Monascus perus KLHS4 at 25 to 30 ° C. for 10 to 20 days, and more preferably at 28 ° C.

The potato-sugar medium is prepared by the addition of agar in the supernatant boiled in water to the supernatant (dextrose) and solid medium (agar) and autoclaved.

When culturing in a solid medium in step 2, after culturing Monascus Perus KLHS4, the solid medium is chopped and extracted with methanol, and when cultivated in a liquid medium after culturing the strain culture medium and the culture mycelium filter medium Was separated using, and the culture supernatant was extracted with methanol to obtain a red dye.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not completed by the examples.

<Example 1> Selection of Monascus perforus strains with high red yeast pigment production capacity

The present inventors analyzed the pigmentation degree of the solid medium in the Monascus perpurus strains in order to select Monascus purpureus strains with high red yeast pigment production capacity. Specifically, the Monascus perforus strains collected in Korea were cultured in a 15 ml potato-sugar-agar solid medium at a temperature of 28 ℃ for 15 days. In the potato-sugar-agar medium, 10 g of dextrose and 15 g of agar were added to 500 ml of supernatant boiled in 1 kg of raw potatoes in 1000 ml of water for 30 minutes so that the total volume was 1000 ml. After fitting, using a high pressure moist heat sterilizer, sterilizing at 15 atm, 121 ° C. for 15 minutes, lowering the temperature to 60 ° C., and then solidifying it by pouring it into a sterile petri dish.

Next, the present inventors compared the degree of pigmentation of the mycelium and solid medium of each hongguk strain cultured in potato-sugar-agar solid medium. In addition, the cultured solid medium was chopped finely to extract the pigment with 100 ml of methanol, the chromaticity was compared to the extracted methanol solution and the absorbance was investigated using a Spectrometer in the range of 300-700 nm. .

As a result, a novel Monascus perforus strain, which is considered to have a high absorbance compared to other red mussels strains and has a high yield of red mussel pigments, was selected. The present inventors named the novel Monascus perperus strain having high red yeast pigment production capacity as " Monascus purpureus KLHS4" and deposited it on the Korea Biotechnology Research Institute Gene Bank on November 19, 2001 ( Accession number: KCTC 18097P)

The dry weight of the mycelium was 120 ± 40 mg when the Monascus perus KLHS4 of the present invention was incubated for 7 days at a temperature of 28 ° C. in 50 ml of potato-sugar liquid medium. After incubation for 7 days by inoculating KLHS4 in potato-sugar-agar solid medium, the mycelial growth was 65 ± 5 mm. Lipolytic, predegradable, proteolytic and beta-glucosidase in solid medium supplemented with various substrates prepared according to Paterson and Bridge, Biochemical Techniques for Filamentous Fungi IMI Technical Handbooks, 1994, 19-31. β-glucosidase) activity was confirmed. The length of hyphae observed under the light microscope was varied and the width was 4 ± 0.5 um. The spores (conidium) was an oval shape separated from the hyphae and the diaphragm in the form of bulging the front end of the hyphae branched from the parent hyphae. Cleistothecium with ascospore was also identified. These shapes were similar to those observed by Carels (Carels and Shepherd, J. Bacteriol , 1975, 122: 288-294). Red pigment was uniformly distributed in the cytoplasm of mycelia. The optical microscopic findings of KLHS4 are similar to those of ATCC16365.

Comparative Example 1 Comparison of Pigment Spectrometers of Monascus Perus KLHS4

The present inventors compared with the Monascus purpureus ATCC16365, which is known to have a high production of red pigment, in order to investigate the pigment spectrometer of the high red pigment production in Monascus pertussis KLHS4 isolated in Example 1. It was. Specifically, 15 ml of potato-sugar-agar solid medium (solid medium prepared as in Example 1) and 50 ml of potato-sugar were prepared using the novel Monascus perus KLHS4 and Monascus perus ATCC16365. After culturing for 15 days in a liquid medium (made of the composition except the agar of the medium production component shown in Example 1) was compared with the production of red pigment.

As a result, Monascus perus KLHS4 secreted the red pigment into a solid medium, the color of the medium was darker red than the control group Monascus Perus ATCC16365 ( Fig. 1 ). In addition, the color of the Monascus Perus KLHS4 mycelium was uniformly red, whereas the control Monascus Perus ATCC16365 was pink.

Therefore, the novel Monascus perus KLHS4 of the present invention is a red yeast strain forming a uniform red mycelium.

<1-1> Comparison of Potato-Sugar-Agar Solid Medium Cultures

The present inventors compared the color of the methanol extract solution after extracting the pigment by slicing finely cultured potato-sugar-agar solid medium in 100 ml of methanol.

As a result, Monascus perus KLHS4 showed a darker red color than the control group ATCC16365 ( A of FIG. 2 ).

Two-fold dilutions of the methanol extracts prepared above were compared and examined for spectrometer profiles in the 300-700 nm range.

As a result, it can be seen that the Monascus perus KLHS4 exhibits about two times higher absorbance than the Monascus perus K. ATHS16365, so that the Monascus perus KLHS4 produces a large amount of pigment ( FIG. 3A ).

In addition, 1 mL of the methanol extract prepared above was concentrated to 20 times, and then smeared onto a thin layer chromatography (TLC) plate and TLC was performed using chloroform-methanol-distilled water (65: 25: 4) as a developing solvent. , TLC pattern of the pigment developed in the above manner was compared under visible light and long wavelength UV.

As a result, the developmental aspect of TLC showed the same size band in Monascus perus KLHS4 and Monascus perus ATCC16365, but yellow (yellow) band with an Rf value of 0.53 only appeared in KLHS4 ( FIG. 4 ). It can be seen that there is a difference between the two strains in the amount of each band, especially in the case of the red band Rf value of 0.84 distinctly more Monascus Perus KLHS4 than the Monascus Perus ATCC16365.

From the above results, it can be seen that in the case of potato-sugar-agar medium cultures, the Monascus perus KLHS4 is a strain that produces a greater amount of pigment than the Monascus perus ATCC16365 strain.

<1-2> Comparison of potato-sugar liquid medium culture

After culturing the strain in potato-sugar liquid medium, the present inventors separated the culture medium and the culture mycelium using a filter paper, and compared the color of the culture supernatant.

As a result, even in the culture supernatant, Monascus Perus KLHS4 showed a more intense color than the Monascus Perus ATCC16365 strain which is a control ( Fig . 2B ).

Comparing the visible light spectrometer aspect of the culture supernatant isolated from the above, according to the absorbance wavelength of the pigment, the Monascus Perus KLHS4 strain showed 2-3 times higher absorbance than the Monascus Perus ATCC16365 ( Figure 3b ).

In addition, the present inventors dried 1 mL of the culture supernatant, dissolved in 50 μl of methanol, and smeared onto a TLC plate to perform TLC using a developing solvent to compare the aspects.

As a result, the pattern of the developed TLC pigment bands was similar between the two strains, but when compared to the amount, the Monascus perus KLHS4 strain produced a greater amount ( FIG. 4 ). This shows that the Monascus perus KLHS4 strain produced more pigment in the same culture supernatant than the Monascus perus ATCC16365 strain and secreted into the culture solution.

From the above results, it is shown that the Monascus perus KLHS4 strain has superior pigment production compared to the control Monascus perus ATCC16365 strain.

<1-3> Comparison of mycelium

The inventors crushed the isolated mycelium using a sonicator and then separated the cytoplasm using a centrifuge. The visible light spectrometer pattern of the solution extracted by adding three-fold methanol to the mycelia and ground cytoplasm by the above method was compared. The absorbance of the spectrometer was 5 or more, so it was not possible to measure it, using the solution diluted 4 times in the case of Monascus Perus KLHS4 strain.

As a result, the pigment absorbance showed similar absorbance in both strains at the 500 nm site, but considering the dilution ratio, the Monascus perus KLHS4 was about 4 times higher in pigment production than the Monascus perus ATCC16365 ( Fig. 3c ) . ).

Particularly, in the case where the pigment absorbance was 400 nm, it was found that the Monascus perus KLHS4 strain had a dye production capacity of about 12 times or more in consideration of the dilution factor compared to the Monascus perus ATCC16365 strain.

In addition, the present inventors dried the separated 1 ml of the cytosol solution and dissolved the dried cytoplasm in 50 μl of methanol to perform TLC by the method described above and compared the aspects.

As a result, the pattern of the TLC developed bands in both strains was similar but the amount was different ( Fig. 4 ). In particular, the yellow (yellow) band with an Rf value of 0.87 and the red bands with an Rf value of 0.84 and 0.76 showed that the Monascus perus KLHS4 was much higher than the Monascus perus ATCC16365 strain.

From the above results, it can be seen that Monascus perus KLHS4 is a strain with a higher amount of pigment production than the Monascus perus ATCC16365 strain.

Comparative Example 2 Antibacterial Activity Test of Monascus Perus KLHS4

The present inventors examined whether Monascus perus KLHS4 secreted an antimicrobial agent and exhibited antimicrobial activity. Specifically, the culture supernatant of each strain to determine the antibacterial activity of the isolated culture supernatant after culturing the Monascus Perus KLHS4 and Monascus Perus ATCC16365 isolated in Example 1 in a potato-sugar liquid culture medium After drying, it was dissolved in 100 μl of distilled water to make a 30-fold concentrate.

In a nutrient medium ( Ba ) coated with Bacillus subtilis (NA), a 100 µl volume was made, and the concentrate of the strain was diluted with distilled water, incubated in an incubator at 37 ° C. for 18 hours, and then Investigate if produced.

As a result, both strains showed antibacterial activity. In particular, Monascus perus KLHS4 showed a 5 mm thick antibacterial ring, whereas Monascus pertussis ATCC16465 showed a 2.5 mm antibacterial ring ( FIG. 5 ).

From the above results, it can be seen that the Monascus Perus KLHS4 strain of the present invention produced more than twice as many antibacterial active substances as compared to the Monascus Perus ATCC16465 erythrocyte strain.

As described above, the Monascus Perus KLHS4 of the present invention produces more pigments than the Monascus Perus ATCC16365 red yeast strain, so it can be provided as a red yeast strain used in the manufacture of fermented foods such as red wine and red rice and processed meat products. It can be used as a production strain of food coloring food additives, such as color correction of, and can be used for the production of antibacterial active material.

Claims (2)

Monascus purpureus KLHS4 (Accession No .: KCTC 18097P) 1) incubating the Monascus perus KLHS4 of claim 1 in potato-sugar solid medium or liquid medium; And 2) a method of producing a red yeast pigment comprising the step of extracting the red yeast pigment using the cultured solid medium or liquid medium using methanol.